Investigation of a Mitochondrial Twin Arginine Transport Pathway in Arabidopsis thaliana

Abstract

Most proteins are synthesized at a location different to their site of function. For example, most of the proteins found in cell organelles such as mitochondria or chloroplasts are encoded by nuclear genes, which are transcribed in the nucleus and translated in the cytoplasm. Cells therefore, must have efficient protein transport processes to allow proteins to cross membranes such as those bounding cell organelles like mitochondria or chloroplasts. Proteins destined for organelles are synthesized as larger molecular weight precursors, containing targeting sequences, which direct the precursor to the appropriate transport apparatus at the membrane.One such vital protein transport pathway is the Tat (Twin Arginine Translocation) pathway. The Tat pathway has been found in a wide variety of organisms including bacteria, archaea and eukaryotic organelles such as the chloroplast. The chloroplast Tat pathway (or analogous Tat pathways in bacteria) is capable of transporting fully folded proteins across membranes using only a proton motive force instead of ATP for energy. It is composed of three protein components, namely cpTatC (TatC), Tha4 (TatA) and Hcf106 (TatB) that work together to allow the transport of precursor proteins containing an obligate twin arginine motif in the targeting sequence.However, recent experiments using Nicotiana tabacum cells showed an up‐regulation of a mitochondrial gene coding for a mtTatC protein when the cells was subjected to salicylic acid treatment, which mimics a defense response in the plant to attack by pests. However, single nuclear genes encode both of the other Tat components. Analogous transport pathways in certain Gram (+) bacteria demonstrate that a minimal functional Tat pathway is composed of TatC and TatA (Tha4 homologue). We have confirmed a similar upregulation under stress of salicylic acid of the putative mitochondrial tatC gene in Arabidopsis thaliana. The presence and upregulation under stress of the mttatC gene suggests the possible presence of a mitochondrial Tat pathway, which would require the dual localization of a TatA homology, such as Tha4.To test the possible dual localization of Tha4 to mitochondrial, we have transformed Arabidopsis thaliana with Tha4‐GFP constructs and looked for its localization in the presence or absence of various biotic or abiotic stresses. In addition, we will look at changes in gene expression using RT‐PCR technique to look for Tha4 upregulation in the presence or absence of the stress.Evaluating the presence of a mitochondrial Tat pathway will shed light on a unique protein sorting and transport mechanism in plant mitochondria, a facility previously thought to be lost during the mitochondrial evolutionary process. Therefore investigating this protein sorting pathway in mitochondria may further our understanding of acquired innate immunity within flowering plant species.